ABSTRACT: The goal of proposed project is to determine the molecular identity of myosin light chain phosphatase phosphatase (MYPT1 phosphatase) and clarify the regulatory role of this poorly investigated critical component to create a molecular and cellular basis for understanding of the physiology and pathophysiology of smooth muscle contraction. Smooth muscle contraction is regulated by the Ca2+ independent pathway in addition to the well known Ca2+ dependent pathway. The key component of the Ca2+ independent pathway is myosin light chain phosphatase (MLCP), whose activity is regulated by the phosphorylation of the regulatory subunit of MLCP, called myosin targeting subunit 1(MYPT1). The research in the past has centered on the RhoA/ROCK pathway, a protein kinase phosphorylating MYPT1. However, recent studies have suggested that the Ca2+ independent regulation of MLC phosphorylation cannot solely be explained by RhoA/ROCK. We propose that MYPT1 phosphatase is the missing regulatory component that explains the unsolved research problem for understanding smooth muscle contractile regulation. Nothing is known about this important regulatory component. Our recent results have suggested that MYPT1 phosphatase is regulated during the contraction-relaxation cycle in smooth muscle (Nakamura et al., 2007). Furthermore, MYPT1 phosphatase is not inhibited by CPI17,which potently inhibits MLCP activity, suggesting that MYPT1 phosphatase is a different molecule from MLCP. Based upon these findings, we propose the following hypothesis. External stimuli alters the MYPT1 phosphatase activity, which causes the change in the MYPT1 phosphorylation level, thus regulates MLCP activity concertedly with the regulation of the RhoA/ROCK pathway. The proposed project will address this hypothesis. First we will isolate MYPT1 phosphatase from smooth muscle and determine the partial amino acid sequence of the subunits of MYPT1 using a Mass Spectrometry technique. Based upon the sequence information, we will identify the genes encoding the MYPT1 phosphatase holoenzyme and functionally express this enzyme (Aim 1). We will then study the characteristics and the regulation of MYPT1 phosphatase at the molecular level. A key question is how MYPT1 phosphatase activity is regulated. We hypothesize that the non-catalytic subunits of MYPT1 phosphatase play a key role in the regulation, and we will study the regulatory function of the non-catalytic subunits including the effect of phosphorylation using Mass Spectrometry analysis (Aim 2). In Specific Aim 3, we will test the effect of elimination of the identified MYPT1 phosphatase on MLCP activity and MLC phosphorylation in smooth muscle to confirm the importance of the identified MYPT1 phosphatase. Finally we will examine the regulation of MYPT1 phosphatase in smooth muscle by external stimuli. It is anticipated that the obtained information of MYPT1 phosphatase will provide a clue to understand the physiology and pathophysiology of organs containing smooth muscle.